The DNA of all eukaryotic cells wraps around histone proteins to form nucleosomes, the basic structure of chromatin. Histones are essential for this packaging of DNA within the chromosomes, and mutation of individual histones in vivo alters the general organization of chromatin throughout the eukaryotic nucleus. A number of posttranslational reactions, such as phosphorylation, acetylation, ADP-ribosylation, and ubiquitination, reversibly modify histones. The acetylation state of histones can affect structural alterations in local chromatin architecture during nuclear processes such as transcription. Furthermore, histone deacetylation plays a pivotal role in controlling access of transcriptional activators and the basal transcription machinery to regulatory sequences in the underlying DNA template, and positively or negatively affects the rate of gene transcription.
The dynamic state of histone acetylation is tightly regulated and maintained by histone acetyltransferase and histone deacetylase enzyme activities. It is widely held that acetylation of core histones destabilizes local nucleosome structure and allows transcription factor access. Conversely, deacetylation causes a tighter association between DNA and histones and favors transcriptional repression and gene silencing (Ayer, Trends in Cell Biology, 1999, 9, 193-198).
Modulation of chromatin architecture by nucleosome remodeling and deacetylating complexes is implicated in control of cell cycle progression, and in both the genesis and the suppression of cancer (DePinho, Nature, 1998, 391, 533, 535-536). Support for this hypothesis comes from the observation that histone deacetylases mediate the function of the chromosomal translocation/gene fusion proteins PML-RARα and PLZF-RARα, and of the frequently mutated tumor suppressor protein RB, contributing to the development of cancers such as acute promyelocytic leukemia (Kouzarides, Current Opinion in Genetics & Development, 1999, 9, 40-48).
Mammalian histone deacetylases 1 and 2, (HDAC1 and HDAC2) have been well characterized and previously found to exist in either of two large, multisubunit protein complexes, called the mSin3 complex (named for one component homologous to the yeast SIN3 gene) and the NuRD complex (nucleosome remodeling and deacetylating). The emerging model of the function of these HDAC-containing complexes is that they are recruited for transcriptional repression under one set of conditions and released and exchanged for histone acetyltransferase coactivators under a different set of conditions. Recently, however, a novel third HDAC1/2-associated complex, distinct from the mSin3 and NuRD complexes, was identified and found to contain a protein called CoREST (You et al., Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 1454-1458).
CoREST (also known as KIAA0071) was identified previously as a corepressor for REST/NRSF (RE1 silencing transcription factor/neural restrictive silencing factor), a transcription factor involved in maintenance of cellular identity by mediating long-term repression of neural-specific genes in non-neural cells. At its C-terminus, CoREST contains two SANT (SWI3/ADA2/NCoR/TFIIIB B″) domains, a structural feature also present in the nuclear receptor corepressor (NCoR)/silencing mediator for retinoic acid and thyroid hormone receptors-extended (SMRTe) proteins (Aasland et al., Trends Biochem. Sci., 1996, 21, 87-88; Andres et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 9873-9878).
The SMRT and NCoR corepressor proteins both form complexes with mSin3 and histone deacetylases to induce local chromatin condensation and mediate transcriptional silencing of important regulators. With the finding that the CoREST protein is also stably associated with HDAC1/2 complexes purified from HeLa cell extract (You et al., Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 1454-1458), it appears that a corepressor family of SANT domain-containing transcriptional repressors regulates important signaling pathways via chromatin remodeling complexes. SANT domain proteins may play a general role in HDAC complex assembly (Humphrey et al., J. Biol. Chem., 2001, 276, 6817-6824), where the composition of the alternative histone deacetylase complexes mediate specific repression pathways (Grimes et al., J. Biol.Chem., 2000, 275, 9461-9467).
A model for the role of different HDAC complexes is that the mSin3-HDAC complex is recruited for simple deacetylation of dynamically regulated promoters, whereas the NuRD and CoREST containing HDAC complexes are recruited to promoters that require stable repression, such as for tissue specific silencing, or for heritable epigenetic states such as genomic imprinting. The associated non-HDAC enzymatic activities may determine the nature of the repression (You et al., Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 1454-1458).
In contrast to REST, which is expressed only in non-neuronal cell lines, CoREST was found to be expressed in all human tissues and all cell lines tested (Andres et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 9873-9878). The CoREST protein is localized to the nucleoplasm and excluded from nucleoli of human skin fibroblasts (Humphrey et al., J. Biol. Chem., 2001, 276, 6817-6824). During development of mouse embryonic tissues, however, the expression pattern of CoREST is restricted. Specifically, in early embryogenesis, mSin3 is widely expressed throughout the embryo, but CoREST expression is only strongly expressed in the head mesenchyme, whereas later in development, this disparity is no longer apparent. This suggests that the composition of the REST repressor complex during development is dynamic and that CoREST may be recruited for more specialized repressor functions (Grimes et al., J. Biol.Chem., 2000, 275, 9461-9467). The CoREST protein is localized to the nucleoplasm and excluded from nucleoli of human skin fibroblasts (Humphrey et al., J. Biol. Chem., 2001, 276, 6817-6824).
The pharmacological modulation of the activity and/or expression components of histone deacetylase/corepressor complexes is believed to be an appropriate point of therapeutic intervention in pathological conditions such as cancer.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of CoREST and investigative strategies aimed at studying CoREST function have involved the use of antibodies for purification and cellular localization studies. Therefore, there exists a long felt need to identify methods of modulating transcriptional repression complexes and specifically for agents capable of effectively modulating CoREST function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of CoREST expression.
The present invention provides compositions and methods for modulating CoREST expression.